Analytical validation of a novel point‐of‐care immunoassay for canine N‐terminal pro‐brain natriuretic peptide analysis

Abstract Background N‐terminal pro‐brain natriuretic peptide (NT‐proBNP) is a widely used point‐of‐care (POC) cardiac biomarker in human medicine. Canine NT‐proBNP is used less in veterinary medicine, possibly due to the lack of a POC canine NT‐proBNP assay resulting in temporal delay, increased degradation in transport, and high reported variability in the available assay. A new quantitative POC analyzer allows fast, onsite measurement of NT‐proBNP, minimizing preanalytical error and reducing variability. Objective We aimed to analytically validate an NT‐proBNP assay (Vcheck) according to American Society of Veterinary Clinical Pathology (ASVCP) and Clinical Laboratory Improvement Amendments (CLIA) specifications. Methods Archived and prospective plasma and serum samples were collected from male and female, client‐owned dogs of various breeds with cardiac abnormalities (n = 81) and a healthy control population (n = 225). Precision, accuracy, analytical sensitivity, and specificity, and other statistical analyses were performed. Results Imprecision was considered acceptable with a coefficient of variation ranging from 9% at 4000 pmol/L to 20% at 600 pmol/L. The lower limit of quantitation was 650 pmol/L based on repetitive measures evaluation. Comparison of the Vcheck assay with the Cardiopet NT‐proBNP assay revealed an excellent correlation with minimal bias when preanalytical factors were controlled. Significant degradation of NT‐proBNP occurred when current methods were used at refrigerated and room temperatures, which could change diagnostic and prognostic decision‐making. Age‐partitioned reference intervals have high reference values of 750 pmol/L and 1280 pmol/L for juvenile and adult dogs, respectively. Conclusions The Vcheck NT‐ProBNP assay provides analytically acceptable results. Onsite testing can minimize variability related to preanalytical error and provide clinically useful contemporaneous results. Samples should be centrifuged immediately and analyzed within 2 hours of collection.


| INTRODUC TI ON
B-type natriuretic peptide (BNP) is a 32-amino acid cardiac natriuretic peptide hormone that is secreted into the circulation by cardiac myocytes and fibroblasts in response to myocardial stress or stretch of the heart's walls due to increased volume and pressure, and its production is therefore significantly upregulated in cardiac failure. BNP binds to receptors in organs such as the kidney, stimulates increased intracellular cGMP production, and induces diuresis, vasodilation, inhibits renin and aldosterone production, and inhibits cardiac and vascular myocyte growth, and possibly inhibits fibroblast proliferation. BNP is secreted as a prohormone, proBNP, and then cleaved into the biologically active hormone, BNP, and the nonactive amino-terminus, NT-proBNP (76 amino acids); therefore, the concentration of either can be used to assess the magnitude of myocardial wall stress or stretch ( Figure 1). 1 However, NT-proBNP is more stable and has a longer half-life than both the prohormone and BNP, making it a useable diagnostic analyte. When antibodies are targeted to the C-terminal end or central regions of NT-proBNP, measured concentrations of NT-proBNP are less susceptible to degradation. 2 For the past two decades, NT-proBNP and other natriuretic peptides have been explored as valuable biomarkers for cardiac disease in both veterinary and human medicine.
In people, NT-proBNP has been thoroughly analytically and diagnostically validated. NT-proBNP is relatively inexpensive, easily and rapidly measured, reflects a pivotal pathophysiologic pathway, provides additional novel information to available clinical data, and has been proven to facilitate the diagnosis, prognosis, and management of human heart failure, thereby approximating an ideal cardiac biomarker. 3 NT-proBNP is currently used as a prognostic indicator in human patients and appears to predict adverse outcomes better than other cardiac biomarkers upon patient follow-up, suggesting the ability to aid in risk stratification. 4 Additionally, a metanalysis of 18 research studies revealed that high NT-proBNP concentrations, indicating cardiac stretch, were associated with a higher risk of death due to acute respiratory distress syndrome for up to 60 days. 5 Thus, NT-proBNP is an important prognosticator for both left heart and right heart overload, which includes either cardiogenic or pulmonogenic etiologies.
In dogs, analytical and diagnostic validation has been investigated for an NT-proBNP ELISA assay 6 (IDEXX, Westbrook, ME, USA). 7,8 There are significant reported differences between human and dog NT-proBNP, including reference values, diagnostic thresholds, and stability. Previously reported canine reference intervals have ranged up to 900 pmol/L in the general adult dog population, 9 with some breed variability in optimized decision thresholds; such F I G U R E 1 Production of NT-proBNP and BNP. proBNP is cleaved by furin to produce the NT-proBNP and BNP molecules when these compounds are released in response to myocardial stretch. From Hall C. Essential biochemistry and physiology of (NT-pro)BNP. Eur J Heart Fail. 2004;6:257-260. doi:10.1016/j.ejheart.2003.12.015. Used with permission. NT-proBNP, N-terminal pro-brain natriuretic peptide as a cutoff value of 457 pmol/L in Doberman pinschers has been reported to accurately identify occult dilated cardiomyopathy, when used in combination with a Holter monitor. 10,11 In contrast, in greyhounds, which also have relatively high cardiac troponin concentrations, the mean NT-proBNP concentration in a population of healthy greyhounds was found to be 946 pmol/L. 12  This study sought to fully characterize analytical performance, including preanalytical and analytical factors, and verify manufacturer's claims for canine NT-proBNP (Bionote, Hwaseong-si, Gyeonggi-do, South Korea) measured using V200 and V2400 Vcheck analyzers (Bionote). This is a novel in-clinic analyzer that minimizes time to analysis so that a more robust diagnostic performance assessment can be completed. Validation was carried out according to  All data regarding history, physical examination, and diagnostic findings were recorded in a standardized data sheet.

| Sample handling, collection, and transport
Sample handling is summarized for the different parts of the verification as follows: Courier and laboratory handling of the samples is unknown.

| Measurement of NT-proBNP
The Vcheck canine NT-proBNP Test kit (Bionote) uses anti-canine NT-proBNP antibodies with conjugated fluorescence microparticles which are released, creating a signal when the antibody binds canine NT-proBNP. The Vcheck 200 and Vcheck 2400 instruments measure final fluorescence in an identical manner, although the 200 is designed for a single dry chemical cartridge and the 2400 has a multi-assay capacity of 24 dry chemical cartridges at one time. All instruments were calibrated using Bionote's recommended software and specifications prior to analysis. Currently, there are no available assayed quality control materials (QCMs) with a concentration in the linear range of the canine assays. The assay was performed in accordance with the manufacturer's instructions. 16 Briefly, patient-side, 100 μL of serum was placed in the supplied diluent tube and mixed by pipetting. The mixture was loaded to the well present in the dry chemical cartridge, which was inserted in the instrument. Results are reported by the instrument after a 15-minute incubation. The LOQ of 500 pmol/L has been set by the manufacturer, and any concentration less than that is reported as <500 pmol/L.
The Cardiopet NT-proBNP (IDEXX) is described as a secondgeneration canine NT-proBNP sandwich ELISA. It includes an increased number of calibrators with increasing concentrations of NT-proBNP peptide in a protein buffer base as well as three controls (low, medium, and high) that are routinely analyzed and provide internal quality control for each assay run. 6 Ethylenediaminetetraacetic acid plasma was recommended as the best sample for Cardiopet NT-proBNP at this time upon phone consultation with IDEXX representatives; although online sampling directions state that serum samples are acceptable, but must be refrigerated. This was presumed to be due to differences in reported instability of plasma vs serum at refrigerated and room temperatures. 6 Therefore, this study submitted plasma samples based on verbal recommendations by the manufacturer that this was a preferred sample type. All plasma samples delivered to IDEXX were transported within the current manufacturer recommendations: and three times the upper reference limit. Methods were compared using linear regression and Bland-Altman plots, with calculated bias and other descriptive statistics. 18 Total analytical error was calculated using modified (2 × CV) + bias. 19 The lower LOQ was verified and modified using an acceptable CV (<20%) in canine serum samples using a repetitive measures study of the target matrix. Reference intervals were calculated according to the guidelines from the ASVCP, using Tukey's outlier analysis with a z < 0.05, the Robust method for <120 reference values, and the recommended nonparametric analysis for datasets with >120 reference values. 20 To assess clinical differences, the number of values with a different classification or diagnosis was divided by the total number of values and multiplied by 100.

| Precision and LOQ
Imprecision varied across concentrations with the lowest CV at the highest concentration as expected ( Table 1) Additionally, the 95% CI of the intercept was much larger, ranging from −278 to 268. The coefficient of determination (R 2 ) decreased to 0.8. The mean difference found using the Bland-Altman analysis was 723 pmol/L ( Figure 3B).

| Observed total error
Bias could not be ascertained in this assay as there is no gold standard method for canine NT-proBNP and the concentration of commercially available QCM (constructed to assess human assays) was below the linear range of the canine assay. Therefore, total error was estimated using 2 × CV. It is expected that the total observed error in the Vcheck NT-proBNP assay will range from approximately    The lower line of the quartile box represents the median (<500 pmol/L) as the majority of the results generated from the juvenile population were below the LOQ of this assay. The 95% medical reference interval is <500-750 pmol/L after three outliers were removed using Tukey's outlier analysis. LOQ, limit of quantitation; NT-proBNP, N-terminal pro-brain natriuretic peptide

≤5 None
Vitamin C ≤1000 None Abbreviation: NT-proBNP, N-terminal pro-brain natriuretic peptide. as reference intervals are higher than this cutoff. As no commercially available, stabilized QCM is available for the assessment of canine NT-proBNP, we recommend that users of this analysis archive frozen aliquots with high-and low-level serum samples such that only one freeze-thaw cycle occurs. These aliquots can be used monthly or periodically to assess analytic precision, especially at low concentrations, to ensure adequate performance.
Accuracy assessment was challenging due to a lack of assayed, commercially available QCM at the needed concentration range found in dogs. Human reference intervals are also age based with high reference limits around 20 pmol/L, which is more than an order of magnitude lower than concentrations found in healthy Therefore, further studies examining the stability of NT-proBNP after freezing are warranted, and researchers and quality assurance specialists using archived samples are urged to assess storage changes at −20°C prior to use. The magnitude of the possible impact of the preanalytical factors on the reported biological variability 25 and the diagnostic performance of canine NT-proBNP concentrations measured by the Cardiopet assay is unknown, but any off-site assay is likely to suffer more from these factors than a POC assay. In addition, the temporal delay in test results can be likewise overcome by a fast point-of-care assay. It should also be noted that sample freezing has been previously reported to result in an increased concentration using some methods. 13 Taken together, the availability of a valid fast POC canine NT-proBNP assay will likely contribute to better diagnostic results without preanalytical error, an increase in clinical use, and a flurry of new diagnostic validation studies.
The analysis of the healthy dog population revealed a significant difference between age categories based on previously published juvenile, adult, and geriatric categorization, which is consistent with that reported in human medicine. 21 The current high reference value (900 pmol/L) used by both manufacturers to define a normal population is not partitioned to a level that is useful diagnostically. Veterinarians play a key role in implementing health screening and improving health care for elderly pets. 21 Cardiac biomarkers that have age-optimized reference intervals, as well as disease and breed targeted decision thresholds, will provide an additional effective diagnostic tool for this endeavor. The adult reference intervals generated in this study are higher than those previously reported by manufacturers, which might be due to the method used, immediate sample processing with no degradation, the reference population, or correctly applied, partitioned statistics in accordance with ASVCP guidelines.

| CON CLUS IONS
This Vcheck validation study found that age-partitioned reference Maeve Heard for technical assistance in processing and spreadsheet organization.

D I SCLOS U R E
This study was funded by Bionote USA, Inc., Big Lake, MN, USA.